Academic Journal of Nawroz University
(Issue : 2)
(Volume : 10)
Molecular Classification of the Earthworm Aporrectodea trapezoids and the Effect of Sodium Nitroprusside and Norepinephrine on the Contractility of Crop and Gizzard Smooth Muscle.
Background: Earthworms are considered as one of the ecologically important invertebrates with highly cryptic genetic diversity. Since the morphological methods of classification are unprecise, it is necessary to classify the species depending on molecular bases using PCR techniques. In addition, studying the effect of some agonists such as Sodium nitroprusside (SNP) and Norepinephrine (NE) on the smooth muscle of earthworm is yet poorly studied.
Objective: This study focused on the molecular classification using the mitochondrial COX 1 gene as a DNA marker. In addition, it also included the effect of nitric oxide (NO) donor sodium nitroprusside (SNP) and norepinephrine (NE) on the contractility of crop and gizzard smooth muscle of the earthworm.
Martial and Methods: For molecular classification of earthworm, DNA was extracted from crop and gizzard amplified using a universal primer (forward primer LCO14900 and specific reverse primer (SPE-CO1-A25) for amplification of the mitochondrial cytochrome oxidase subunit I (COX1) gene using touchdown PCR technique. For physiological studies, the contractility of crop and gizzard smooth muscles of earthworm was studied using an organ tissue bath containing ringer solution and recording the contraction isometrically. Also, the effect of different doses of SNP and NE on the contractility of the crop and gizzard was studied.
Results: The outcomes demonstrated that universal primers used for the amplification of 523 bps fragment by using Touch down Polymerase Chain Reaction (TD-PCR) identified the species of the earthworm as Aporrectodea trapezoids. The physiological results showed that different doses of norepinephrine (1*10-8 to 1*10-3 M) significantly reduced the contraction of crop and gizzard smooth muscles of the earthworm. While treatment with different doses of SNP (1*10-6 to 1*10-3 M) showed a non-significant (P >0.05) relaxing effect.
Conclusion: The obtained sequence identified the species of earthworm as Aporrectodea trapezoids, it is registered and deposited in the GenBank under accession number (MW354675). Phylogenic analysis of Aporrectodea trapezoids using cox1 indicated its relationship to other strains from the USA. Physiologically, Norepinephrine exerts an inhibitory effect on the Smooth Muscle cell contractility, whereas, SNP produced no effect on the amplitude of the contractility of the crop and gizzard SMCs.
2021-06
Thesis
2021-05-24
Molecular Identification and the Effect of Some Agonists on the Contractility of Crop and Gizzard Smooth Muscle in the Earthworm Aporrectodea trapezoids (Duges, 1828)
The present study was carried out on the earthworms Aporrectodea trapezoids collected from Bezeh-Betas (Zakho City). The study aimed to classify the earthworm species using molecular tools and focusing on the effect of some agonists on the contractility responses of crop and gizzard smooth muscle cells. The molecular classification was performed using mitochondrial COX 1 gene as a DNA marker, and the DNA was extracted from the crop and gizzard tissues of the earthworm and amplified using universal primers (forward primer LCO14900 and specific reverse primer SPE-CO1-A25), and Touchdown Polymerase Chain Reaction T-PCR has used the size of the band obtained from the amplification of COX1 gene was 523 bps. Out of 45 PCR products, five were sent to Macrogen Company (South Korea) for DNA sequencing. The sequencing results were submitted to BioEdite program for sequence alignments. The obtained sequence confirmed that the species is Aporrectodea trapezoids, and this sequence was deposited in GeneBank under the accession number (MW354675). Phylogenic analysis of Aporrectodea trapezoids COX1 indicated its relationship to other strains from the USA.
Regarding the response of crop and gizzard smooth muscle cells to some agonists, such as Hydrogen sulfide, Nicotine, Sodium nitroprusside, and Norepinephrine was recorded. The isolated crop and gizzard smooth muscle isolated from earthworm were fixed in Panlab multi-chamber organ bath system containing ringer solution for recording the amplitude and frequency contractions of crop and gizzard. The crop and gizzard were left in Ringer solutions for 20 minutes for tissue stabilization, followed by its exposure to different doses of Sodium Nitroprusside (1X10-6 to 1X10-3 M), Norepinephrine (1X10-8 to 1X10-3 M), Nicotine (1X10-8 to 1X10-5 M), and Hydrogen sulfide (1-5 mM) then half-maximal inhibitory concentration (IC50) were determined for crop and gizzard
smooth muscle. The obtained results revealed that different doses of norepinephrine, Hydrogen sulfide, and nicotine significantly reduced the amplitude and frequency of contraction of crop and gizzard smooth muscle, which indicates that norepinephrine, Hydrogen sulfide, and nicotine are acting as inhibiting agents. On the other hand, treatment with different doses of sodium nitroprusside showed the non-significant effect on the amplitude and frequency of contraction of crop and gizzard smooth muscle of earthworm.
